Ordered replication of the eukaryotic genome requires that all 10,000-100,000 units of replication (replicons) are duplicated once, but only once, within each cell cycle. To study the mechanisms of replication control in eukaryotic cells we have used as a model system a chimeric replicon derived from the DNA of the tumor viruses SV40 and bovine papilloma virus. In this specific model we have shown that DNA replication is regulated by an interplay between positive factors that are necessary to initiate DNA synthesis and negative elements that prevent re-replication of DNA sequences within a single S phase. The goal of the proposed research is to understand the mechanisms of the negative controls on DNA replication. To understand the mechanisms that negatively regulate DNA replication (i.e. the mechanisms that limit the duplication of any genomic DNA to just once per cell cycle) we will continue to study, both in vivo and in vitro, the cis and trans acting replication control elements we have defined within the bovine papillomavirus genome. We propose to define the minimal cis acting sites within BPV that are necessary for regulated DNA replication, to study the spacing requirements for these two cis-acting sequences, and to investigate the interaction between these sites and the transacting regulatory E1 protein, by both genetic and physical approaches. We will begin to analyse the biochemical mechanism involved in replication control by establishing an in vitro system in which the regulated replication of SV40-BPV chimeric minichromosomes can be studied directly. In an extension of these experiments we will determine whether the cell uses similar cis and trans acting elements to regulate the replication of its genomic DNA.